During the past decades, one major focus of heart developmental biology is on the cardiac transcriptional factors (such as Nkx2.5, Isl1, Tbx20), which are conserved coding genes across multiple species and play key roles in controlling cardiogenesis. However, human heart has different size, electrophysiological properties and distinct cardiogenesis process from rodents. Why such species-specific differences exist and what gene programs control human-specific cardiogenesis and heart function are still remaining unknown. Recently, accumulating evidences showed that long noncoding RNAs (lncRNAs) play important roles in cell fate specification and organ development. LncRNAs are > 200 nucleotides noncoding transcripts with limited coding potential. LncRNAs account for ~40% of human transcriptome. Particularly, many lncRNAs are tissue- specific and species-specific with poor interspecies conservations. Additionally, recent evidences demonstrated dys-regulated lncRNAs could cause various cardiovascular diseases. All these evidences allow us to hypothesize that lncRNAs are species-specific genes, which could regulate human-specific cardiogenesis. In our preliminary studies, we recapitulated early human heart development using human embryonic stem (ES) cells. By using whole transcriptome sequencing, we identified hundreds of previously unannotated novel human lncRNAs, which showed lineage-specific expression patterns in human ES cell-derived multipotential cardiovascular progenitor cells (MCPs), cardiomyocytes (CMs), smooth muscle cells (SMs) or endothelial cells (ECs). To date, full sequences and functions of those novel human cardiac- specific lncRNAs are still unknown. Therefore, in this proposal, we will conduct a screening to identify functional novel human lncRNAs, which regulate MCP formation and/or commitment. Particularly, 21 unannotated lncRNAs, which are specifically enriched in human heart progenitor cells (MCPs) with high abundance, were chosen as target lncRNA candidates. Given that some lncRNAs are partially overlapping with host or neighbor genes, complete deletion of lncRNA using classic CRISPR/Cas-9 may partially delete its host/neighbor genes. Thus, we decided to utilize a recently reported inducible CRISPR/Cas-9 interference (CRISPRi) system to knock down lncRNAs without disturbing their host/neighbor genes. Meanwhile, an inducible CRISPR/Cas-9 activation (CRISPRa) system will be utilized in this proposal to activate endogenous lncRNA expression. Overall, by taking advantages of our in vitro model of human cardiogenesis in ES cells and the cutting-edge technologies of CRISPRi/a, this proposal aims to define sequences and determine functions of a list of novel human lncRNAs in regulating early human heart development.